The present invention relates generally to an automated cytopathology screening instrument and, more particularly, to a flow cytofluorometer for simultaneously obtaining multidimensional slit-scan type flourescence contours of particles, particularly biological cells, in flow. Various aspects of this invention are an improvement of the invention to which commonly-assigned application Ser. No. 030,880, filed Apr. 17, 1979, by David B. Kay, Leon L. Wheeless, Jr. and James L. Cambier, and entitled "MULTIDIMENSIONAL SLIT-SCAN FLOW SYSTEM" is directed, now U.S. Pat. No. 4,293,221. The entire disclosure of said application U.S. Pat. No. 4,293,221 is hereby expressly incorporated by reference.
A number of approaches have been developed directed to the problem of automating the process of analyzing cells from biological specimens. One particular, but not limiting, purpose is automated prescreening for gynecologic cancer and its precursors. Conditions for a successful system are: (1) a low specimen false negative rate; and (2) an acceptable specimen false positive rate. Achievement of these conditions requires a low single-cell false alarm rate. These functional considerations should preferably be satisfied in a cellular screening system which has a high cell throughput, and which minimizes the amount of complex computation in the analysis required. This basically translates to a question of system resolution.
At one end of the resolution scale are high-resolution systems, for example utilizing a sub-micron scanning spot, wherein a full two-dimensional image of each cell is acquired and processed by a computer. This can involve relatively long data processing and computation times, and consequent low throughput. Particular examples of high-resolution systems are disclosed in the Ehrlich et al. U.S. Pat. No. 3,699,336 and in the Holm U.S. Pat. No. 3,918,812.
At the other end of the resolution scale are low-resolution systems wherein excitation and measuring apertures are larger than the cell of interest and a gross characteristic of each cell is examined, for example total fluorescence at a particular wave-length or light scatter at a particular angle. These systems permit measurements to be made at rates up to several thousand cells per second, and provide valuable information on specific cellular substances and parameter profiles on large populations of cells. However, they have thus far failed to demonstrate a capability to provide sufficient cellular information for the decisions required for application as a screening instrument.
A number of approaches have been proposed for low-resolution systems. The Coulter U.S. Pat. No. 2,656,508 describes the Coulter sensing principle which provides an indication of cell size. Of particular interest with respect to the present invention are the low-resolution systems of the following U.S. patents, which systems generally optically view biological cells from two orientations or simultaneously examine a plurality of optical characteristics: Friedman et al. U.S. Pat. Nos. 3,705,771, 3,785,735, and 3,788,744; and Fulwyler et al. U.S. Pat. No. 3,710,933. As a particular example, the low-resolution system disclosed in the '933 Fulwyler et al patent includes a flow chamber through which cells in suspension flow sequentially in a stream, and a light beam which intersects the cell stream at right angles. The Fulwyler et al. system measures small angle light scatter in one direction, and cellular fluorescence in another.
One particularly promising medium-resolution measurement technique suitable for use in the field of automated cytology is a slit-scan technique invented by L. L. Wheeless, Jr., and S. F. Patten, Jr. This technique is initially described in the Wheeless, Jr., et al. U.S. Pat. No. 3,657,537; and in the literature reference: L. L. Wheeless, Jr., and S. F. Patten, Jr., "Slit-Scan Cytofluorometry", Acta Cytol., Vol. 17, No. 4, pp. 333-339 (1973). Both static cell and flow cytofluorometers are described.
The slit-scan technique is further described, with additional details concerning flow cytofluorometers in particular, in the following representative literature references: L. L. Wheeless, Jr., J. A. Hardy and N. Balasubramanian, "Slit-Scan Flow System for Automated Cytopathology", Acta Cytol., Vol. 19, No. 1, pp. 45-52 (1975); L. L. Wheeless, Jr., D. B. Kay, M. A. Cambier, J. L. Cambier and S. F. Patten, Jr., "Imaging Systems for Correlation of False Alarms in Flow", J. Histochem. Cytochem., Vol. 25, No. 7, pp. 864-869 (1977); and D. B. Kay, J. L. Cambier and L. L. Wheeless, Jr., "Imaging System for Correlating Fluorescence Cell Measurements in Flow", Proceedings of the Society of Photo-Optical Instrumentation Engineers, San Diego, Calif., Clever Optics, Vol 126, pp. 132-139 (Aug. 25-26, 1977). The entire disclosures of each of the references identified in this and the preceeding paragraph are hereby expressly incorporated herein by reference.
The slit-scan technique provides a more complete set of cellular parameters than is available with a low-resolution optical system, without producing the large data matrix associated with a high-resolution system. It represents a compromise solution to the problem of throughput and resolution.
In particular, the slit-scan configuration sequentially records the secondary fluorescence of an elongated portion of a cell (generally transversing the width of the cell) at discrete time intervals as that cell moves relative to a slit-producing aperture, and a sequential series of planar cell volumes are defined by the slit-producing aperture as the cell relatively moves. This type of medium-resolution slit-scan provides a graphic fluorescence contour, which in essence is a plot of the averaged fluorescence along the cell. From such contours, the fluorescence from the nuclei of the cells is readily distinguishable from cytoplasmic fluorescence.
Two general approaches to implementing this concept have been proposed, as well as a hybrid approach. In the first approach, the slit is defined by optical imaging. In particular, the entire cell, or at least a substantial volume, is illuminated by excitation light, and a slit region only of the illuminated volume is viewed, for example through an optical system having a slit field stop or aperture in an image plane, such that only the secondary fluorescence from a defined planar region reaches a detector.
In the second general approach, the slit is defined by the excitation illumination, and slit-imaging is not required. Only a planar volume of the cell is excited to fluorescence, with other cellular regions remaining dark. As typically implemented in a flow system, secondary fluorescence from a cell is recorded as the cell flows through a thin "wall" of excitation illumination.
In a hybrid approach, only a planar region is excited by a slit of excitation illumination, and only the excited planar region is viewed through a suitable slit-imaging system.
In any event, the slit-producing aperture, however defined, ideally is much smaller than the diameter of the cell of interest.
The information from such a slit-scan fluorescence contour has been shown to be quite useful in prescreening. Specifically, for each cell, such characteristics as nuclear fluorescence, nuclear diameter, cytoplasmic diameter, cytoplasmic fluorescence, and nuclear-to-cytoplasmic diameter ratio (N/C ratio) may be determined. This technique is becoming increasingly useful since a comparison data base has been developed to enable the recognition of abnormal cells. In addition to the Wheeless and Patten "Slit-Scan Cytofluorometry" article identified above, the following literature references provide background information concerning the usefulness of the slit scan technique: L. L. Wheeless, Jr., and S. F. Patten, Jr., "Slit-Scan Cytofluorometry: Basis for an Automated Cytopathology Prescreening System", Acta Cytol., Vol. 17, No. 5, pp. 391-394 (1973); L. L. Wheeless, Jr., S. F. Patten Jr., and M. A. Cambier, "Slit-Scan Cytofluorometry: Data Base for Automated Cytopathology", Acta Cytol., Vol. 19, No. 5, pp. 460-464 (1975); and M. A. Cambier, W. J. Christy, L. L. Wheeless, Jr. and I. N. Frank, "Slit-Scan Cytofluorometry Basis for Automated Prescreening of Urinary Tract Cytology", J. Histochem. Cytochem., Vol. 24, No. 1, pp. 305-307 (1976).
In particular, from the Wheeless, Jr., Patten, Jr., and Cambier article entitled "Slit-Scan Cytofluorometry: Data Base for Automated Cytopathology", extensive investigation has shown that nuclear fluorescence from cells stained with Acridine Orange is higher in abnormal cells than in normal cells. The Cambier, Christy, Wheeless, Jr., and Frank article entitled "Slit-Scan Cytofluorometry Basis for Automated Prescreening of Urinary Tract Cytology", demonstrates potential usefulness in a slightly different area.
More recent slit-scan instruments utilize a flow cyto-fluorometer, details of which are described in: L. L. Wheeless, Jr., A. Hardy and N. Balasubramanian, "Slit-Scan Flow System for Automated Cytopathology", Acta Cytol., Vol. 19, No. 1, pp. 45-52 (1975), also referenced above. This flow cytofluorometer implements the hybrid approach to defining a slit-producing aperture. In particular, secondary fluorescence is recorded from a cell as it flows through a thin "wall" of excitation illumination providing slit-excitation, and in addition slit-imaging is employed such that only the excited planar region is slit-imaged to a detector.
In this flow system, relatively transparent, fluorochrome stained cells in suspension ideally flow one-by-one through a focused slit of laser excitation light. Assuming the axis of flow is defined as the Z axis, in essence a planar sheet of excitation light in the X-Y plane is generated defining an excitation region. As the fluorochrome stained cells flow through the excitation region, fluorescence emissions are generated at the intersection of the planar excitation region and the cell. As the cell flows through the excitation region, a plurality of substantially planar parallel cross-sections of the cell along the Z axis are excited to secondary fluorescence. Monitoring the fluorescense emissions generates a slit-scan type contour along the Z axis.
It should be noted that one difference between systems wherein the slit is defined by optical imaging, and systems wherein the slit is defined by excitation illumination, relates to the difference in the shape of the aperture effective cross-section. In the optically-imaged slit-scan system, the aperture is a true slit. In the slit excitation system, the sheet of laser light has a Gaussian intensity distribution in the direction of flow (along the Z axis), and is elliptical in cross-section (in the X-Y plane). This difference, however, is insubstantial in practice, and the expression "slit-scan type contour" as employed herein is intended to refer interchangeably to contours generated by either of these approaches, and both are in fact employed at the same time in one embodiment of the present invention.
One particular problem in evaluating cells by means of a one-dimensional slit-scan type contour along a single axis is that false alarms may occur as a result of such factors as cell orientation and cell overlap. It has been determined that the majority of false alarms are due to either improper cell orientation, multinucleate cells, or overlapping cells oriented such that both nuclei enter the measurement region simultaneously. Such a cell or cells may be completely normal but, in particular orientations, process to greater nuclear fluorescence than uninuclear cells of the same cell type, and thus be erroneously classified as abnormal. Similarly, the entire cell may be oriented with the plane of the cell parallel to the plane of the slit excitation such that there is substantially no discrimination between cytoplasmic fluorescence and nuclear fluorescence. The problem of binucleate cells is discussed in particular in the literature reference: J. L. Cambier and L. L. Wheeless, Jr., "The Binucleate Cell: Implications for Automated Cytopathology", Acta Cytol., Vol. 19, No. 3, pp. 281-285 (1975).
At least four general approaches to solving cell classification problems resulting from particular cellular orientations have been proposed. A first technique is the use of flow nozzles and analysis chambers which tend to produce a desired orientation of the cells. This general approach is suggested by the Hogg U.S. Pat. No. Re. 29,141, and in the literature reference: D. Kay and L. L. Wheeless, Jr., "Experimental Findings on Gynecologic Cell Orientation and dynamics for Three Flow Nozzle Geometries", J. Histochem. Cytochem., Vol. 25, No. 7, pp. 870-874 (1977).
A second technique is to provide a second analysis stage which analyzes cells for which a positive event has been recorded as a result of passage through a first slit-scan type measurement stage. The second stage may employ a higher resolution analysis, and may obtain and analyze a two dimensional image. Such techniques are generally described in the following two literature references: L. L. Wheeless, Jr., D. B. Kay, M. A. Cambier, L. Cambier and S. F. Patten, Jr., "Imaging Systems for Correlation of False Alarms in Flow", J. Histochem. Cytochem., Vol. 25, No. 7, pp. 864-869 (1977); and D. B. Kay, L. Cambier and L. L. Wheeless, Jr., "Imaging System for Correlating Fluorescence Cell Measurements in Flow", Proceedings of the Society of Photo-Optical Instrumentation Engineers, San Diego, Calif., Vol. 126, Clever Optics, pp. 132-139 (Aug. 25-26, 1977).
Third, and particularly in the context of a second analysis stage as mentioned immediately above, it has been suggested that a segmented slit technique would be useful. (Wheeless, Kay, Cambier, and Patten, "Imaging Systems for Correlation of False Alarms in Flow", above.) This technique produces a plurality of slit-scan type contours, each representing fluorescence across only a portion of the cell, and may be considered a low-resolution form of two-dimensional imagery.
Fourth, it has been recognized that apparatus which would provide one-dimensional slit-scan contours along three orthogonal axes would be quite useful in providing additional information useful in reducing the incidence of false alarms, particularly those resulting from binucleate or overlapping cells which are oriented such that, in a single slit-scan type contour, each appears to be an abnormal cell having a high nuclear fluorescence, rather than a binucleate cell or a pair of overlapping cells producing two distinct peaks on the slit-scan contour. In a multi-dimensional slit-scan flow system, in which three orthogonal projections of cell fluorescence are collected, overlapping cells produce three slit-scan contours. In at least one of these contours the nuclei will in most cases be evidenced by two distinct fluorescence peaks, which can be recognized as such, and the nuclear fluorescence measurement disregarded. The Cambier and Wheeless, Jr. article entitled "The Binucleate Cell: Implications for Automatic Cytopathology", cited above, itself refers to the desirability of a system employing three orthogonal slits. An additional such reference is in the Wheeless, Jr., Hardy and Balasubramanian article, also cited above, which describes a "Slit-Scan Flow System for Automated Cytopathology". A more recent analysis of false alarms may be found in L. L. Wheeless, Jr., J. L. Cambier, M. A. Cambier, D. B. Kay, L. L. Wightman and S. F. Patten, Jr., "False Alarms in a Slit-Scan Flow Systems: Causes and Occurrence Rates Implications and Potential Solutions", J. Histochem. Cytochem., Vol. 27, No. 1, pp. 596-599 (1979).
The above-cited Kay, Wheeless, Jr. and Cambier U.S. Pat. No. 4,293,221 discloses various embodiments for obtaining multi-dimensional slit-scan type contours of biological cells in flow. In addition, similar multi-dimensional slit-scan flow systems are disclosed in the following literature references: J. L. Cambier, D. B. Kay and L. L. Wheeless, Jr., "A Multi-Dimensional Slit-Scan Flow System", J. Histochem. Cytochem., Vol. 27, No. 1, pp. 321-324 (1979); D. B. Kay, J. L. Cambier and L. L. Wheeless, Jr., "Imaging in Flow", J. Histochem. Cytochem., Vol. 27, No. 1, pp. 329-334 (1979); J. L. Cambier and L. L. Wheeless, Jr., "Predicted Performance of Single- versus Multiple-Slit Flow Systems", J. Histochem. Cytochem., Vol. 27, No. 1, pp. 334-341 (1979); and L. L. Wheeless, Jr., J. L. Cambier, M. A. Cambier, D. B. Kay, L. L. Wightman and S. F. Patten, Jr., "False Alarms in a Slit-Scan Flow System: Causes and Occurrence Rates Implications and Potential Solutions", J. Histochem. Cytochem., Vol. 27, No. 1, pp. 596-599 (1979).
In several embodiments, three (X, Y and Z axis) orthogonal one-dimensional projections of cell fluorescence are obtained. In one broad approach described in the above-cited Kay, Wheeless, Jr. and Cambier U.S. Pat. No. 4,293,221, fluorochrome stained cells in suspension flow one-by-one through a focused slit of laser excitation light. If the axis of flow is defined as the Z axis, in essence a planar sheet of excitation light in the X-Y plane is generated defining an excitation region. As the fluorochrome stained cells flow through the excitation region, fluorescence emissions are generated at the intersection of the planar excitation region and the cell. By monitoring the fluorescence emissions as the cell flows through the excitation regions, a plurality of substantially planar parallel cross-sections of the cell along the Z axis are excited to fluorescence, and a slit-scan type contour along the Z axis is generated. To generate slit-scan type contours along the cellular X and Y axes, various optical system embodiments, including side imaging and on-axis imaging approaches, effectively define various combinations of cellular linear portions within the Z axis cross-section. The fluorescence or other emissions from these cellular linear portions are then combined by employing an integration technique to generate the desired contours.
In another broad approach described in the above-cited Kay, Wheeless, Jr. and Cambier U.S. Pat. No. 4,293,221 with particular reference to FIGS. 15, 16 and 17 thereof, and one which is particularly relevant with respect to the present invention, a three-dimensional volume of a cell in flow is illuminated with exciting radiation which is not a narrow beam. Three slit-imaging optical systems view a central region in the flow stream, which central region is within the excited volume. The optical systems have three respective orthogonal axes symmetrically located about the stream and directed downward at an angle towards the axis of flow such that the axis of flow forms equal angles with each optical axis. Geometrically, it can be shown that each of the optical axis is angled with respect to the flow or Z-axis at an angle equal to arc tangent 1/.sqroot.2, which is approximately 35.degree.. A slit field stop is employed in the image plane of each optical system, and each of the three slits is oriented to be parallel to each of the three planes respectively of an X'-Y'-Z' coordinate system, so-termed because it is rotated with respect to the flow or Z-axis and with respect to the X-Y-Z coordinate system. Behind each slit field stop, a photomultiplier tube detects the imaged cell fluorescence through a suitable optical wavelength filter, and each slit thus provides a scanned signal spatially orthogonal to the others.
In operation, as a cell flows through the illuminated central region it in effect flows simultaneously through three slit regions. The output of each of the three photomultiplier tubes provides a slit-scan signal spatially orthogonal to the others. In contrast to the embodiments wherein each cell flows through a thin "wall" of excitation illumination, the entire cell is illuminated at once (or at least the volume required for all three slit regions), and the slit apertures are exclusively the result of imaging by the optical systems.
As described in the above-cited Kay, Wheeless, Jr. and Cambier U.S. Pat. No. 4,293,221 an advantage of this system is the simpler signal processing required, as no integration is required and all three slit-scan contours are generated in real time. A disadvantage is that lower resolution is achieved because a greater depth of focus is required (increased by approximately .sqroot.2 compared to systems employing a planar sheet of laser excitation light and various optical systems for defining cellular linear portions). A further disadvantage is a lower signal-to-noise ratio due to lower numerical aperture optics required by the greater depth of focus, as is explained more fully hereinafter.